The goal of this proposal is to develop an effective gene therapy strategy to treat hemophilia A (HemA) by directly transferring lentiviral vectors encoding factor VIII (FVIII) gene into bone marrow cells. Current treatment of HemA patients involves repeated infusions of FVIII proteins which is both costly and inconvenient. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Gene therapy treatment that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired. Hematopoietic stem cells (HSCs) in the bone marrow are an ideal target for gene- and cell- based therapy of genetic diseases, because they are self-renewal and can differentiate into mature blood cells. Intra-bone marrow injection of lentiviral vectors has been shown to effectively transduce bone marrow cells in mice without pre-conditioning. This approach avoids the difficulties encountered by ex vivo HSC gene transfer such as maintenance of stem cell properties, the loss of engraftment potential after cell transfer, and potential cytokine stimulation. Furthermore, no in vitro manipulation of stem cells and pre-conditioning of the subject will be needed using this approach. In order to reduce the potential of anti-FVIII antibody formation, we will incorporate a human FVIII cDNA encoding a less immunogenic B-domain FVIII variant into the lentiviral vectors. Two lentiviral vectors driven by two different promoters, a ubiquitous human elongation factor - 1? (EF1?) promoter (E-LV) and a human megakaryocytic-specific glycoprotein 1b? (GP1b?) promoter (G-LV) have been constructed. FVIII gene expression, correction of HemA phenotype and generation of anti-FVIII immune responses will be evaluated and compared following intra-bone marrow injection of these two lentiviral vectors in hemophilia A (HemA) mice. We will investigate whether low levels of FVIII gene expression in platelets in mice treated with G-LV or high levels of circulatory FVIII in plasma in mice treated with E-LV with or without immunomodulation will be effective to correct HemA phenotype for long-term in mice. We will test the hypotheses that: 1) Following intra-bone marrow injection of lentiviral vectors in unconditioned mice, significant bone marrow cells including primitive HSCs will be transduced and matured into FVIII-expressing blood cells; 2) lentiviral vectors driven by a ubiquitous promoter will direct FVIII or GFP gene expression in a variety of matured blood cells, whereas lentiviral vectors driven by a platelet-specific promoter will direct FVIII gene expression only in megakaryocytes and platelets. 3) Long-term phenotypic correction of hemophilia A will be achieved using intra-bone marrow injection of lentiviral vectors driven by either ubiquitous or platelet-specific promoters with or without immunomodulation. PUBLIC HEALTH RELEVANCE: Our goal of this project is to develop an effective gene therapy strategy to treat hemophilia A. Hematopoietic stem cell gene therapy. Hematopoietic stem cells (HSCs) in the bone marrow are an ideal target for gene- and cell-based therapy of genetic diseases. We will develop a new novel strategy to directly transfer lentiviral or foamy viral vectors into bone marrow cells. This strategy can avoid many difficulties encountered by ex vivo HSC gene transfer. No in vitro manipulation of stem cells and pre-conditioning of the subject will be needed using this approach. In vivo HSC gene therapy may provide long-term therapeutic benefit using either local delivery of FVIII via platelets or ubiquitous delivery of FVIII in plasma with or without immunomodulation.